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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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Reference: A: Determination of the respiration due to the alternative oxidase pathway Bioblast pdf »Versions

O2k-Application: O2

SUIT-category: ce
SUIT protocol pattern: linear ce1;ce2KCN;ce3SHAM

The SUIT-022 is designed to provide an estimation of the mitochondrial respiration in cells due to the alternative oxidase pathway, which is cyanide-and antimycin A-resistant. This protocol must be complemented with the study in parallel of the mitochondrial respiration due to the cytochrome c oxidase (CIV) pathway (see SUIT-023).

Communicated by Huete-Ortega M and Gnaiger E (last update 2019-04-09)

Specific SUIT protocols

SUIT-022 O2 ce D051

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png D051 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2KCN ROUTINE or ROX ce1;ce2KCN
  • Cyanide (usually added as KCN) is a competitive inhibitor of cytochrome c oxidase (CIV). In the presence of an active alternative oxidase (AOX) pathway, after the inhibition of CIV, cells remain in the physiological coupling state ROUTINE. In those occasions where the AOX is not active, after the inhibition of CIV, the (ROX state) is normally achieved, where Rox is the residual oxygen consumption due to oxidative side reactions estimated either after inhibition of CIII, CIV or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration. Cyanide inhibition is reversed by pyruvate and high oxygen levels.
ce3SHAM ROX ce1;ce2KCN;ce3SHAM


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Strengths and limitations

+ This protocol can be used with many type of cells, although optimal concentrations of inhibitors have to be optimised.
+ Short duration of experiment
- Estimation of the linear coupling control is not done
- The use of cyanide requires a careful and in deep cleaning of the chambers after use and careful handling of the chemical to guarantee health safety.

Compare SUIT protocols


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry